Fatty Acid Composition of Lipoprotein Lipids in Hepatobiliary Diseases

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Fatty Acid Composition of Lipoprotein Lipids in Hepatobiliary Diseases

¹

)

Maria Isabel Arranz\ Migi4el Angel Lasuncion2, Jose Perales3, Emilio Herrera2, Isabel Lorenzo1, Cristina Carcamo1, Luis Concostrina1, Juan Villar1 and Rafael Gasalla1

1 Servicio de Bioquimica Clinica

2 Servicio de Bioquimica de Investigation

3 Servicio de Medicina Interna

Hospital "Ramon y Cajal", Madrid, Spain

Summary: Liver damage and alterations in the exocrine function of the gland lead to a profound alteration of the plasma lipoprotein profile. To determine whether hepatic disease results in changes in the lipoprotein fatty acid composition, i. e. to determine whether liver function influences the homeostasis of complex lipids in plasma, we studied the fatty acid profile of lipids from VLDL, LDL and HDL, as well as from total plasma, in thirty-one patients of both sexes with hepatobiliary pathology (compensated liver cirrhosis, uncompensated liver cirrhosis, primary biliary cirrhosis, other intrahepatic cholestasis, and acute viral hepatitis). We also studied a group of healthy adults as controls. We present the lipoprotein profile and the fatty acid composition (myristic C14, palmitic C16, palmitoleic C16 : 1, stearic CIS, oleic CIS : 1, linoleic CIS : 2, eicosatrienoic C20 : 3co6 and arachidonic C20 : 4) of lipoprotein and total plasma triacylglycerols, cholesteryl esters and phospholipids. The main observation of this study is that, despite the profound changes in the lipoprotein profile and the lower abundance of polyunsaturated fatty acids in complex lipids, the composition of all triagylglycerols, cholesteryl esters and phospholipids is very similar for the corresponding lipoproteins of patients with hepatobiliary disease and of control subjects.

This indicates that in the controls as in the studied patients, the exchange of lipids between plasmatic lipoproteins is very rapid and demonstrates the possible importance of the extrahepatic synthesis of cholesteryl ester transfer protein.

Introduction

The liver has a central role in the synthesis of polyunsaturated fatty acids and in lipoprotein metabolism. It is site of synthesis of the greater part of arachidonic acid, cholesterol, triacylglycerols, phospholipids and apoprot- eins that circulate in plasma as lipoproteins. Liver also produces and secretes lecithin : cholesterol acyltransfer- ase2), a plasmatic enzyme that esterifies the cholesterol collected from the cells by HDL (1), and cholesteryl ester transfer protein which allows the exchange of neutral lipids between the different classes of lipoproteins in plasma (2). Liver damage and alterations in the exocrine function of the gland lead to a profound alteration of the plasma lipid profile, modifying the lipoprotein content of total and esterified cholesterol, triacylglycerols, phospholipids (3) and apolipoproteins (4), as well as the activities of lecithin : cholesterol acyltransferase (5), cholesteryl ester transfer protein (6,7) and hepatic lipase

!) This work was supported in part by the Fondo de Investigaci- ones Sanitarias (94/0484), Spain.

2) Enzymes

Alkaline phosphatase (EC 3-1.3.1), Aspartate aminotransferase (EC 2.6.1.1), γ-Glutamyl transferase (EC 2.3.2.2), Lecithin : cholesterol acyltransferase (EC 2.3.1.43).

(8). Liver damage also influences the transformations of polyunsaturated fatty acids (9).

The fatty acid composition of plasma lipids is a reflec- tion of the type of fatty acids in the diet, and of the synthesis and transformation of fatty acids in the organ- ism. The different classes of complex lipids — triacylglycerols, cholesteryl esters and phospholipids — have different compositions of fatty acids. The fatty acid composition of these complex lipids in the different lipoproteins has been studied in detail in healthy subjects, and shown to be similar in VLDL, LDL and HDL (10—

11), thus confirming the rapid exchange of these lipids between these lipoproteins, mediated by the transfer proteins. In contrast, in animal species that lack cholesteryl ester transfer protein, such as the rat and the pig, the fatty acid profile of cholesteryl esters in the distinct lipoproteins is different (12-13).

In patients with liver diseases, especially those with cirrhosis, the fatty acid composition of triacyclglycerols, cholesteryl esters and phospholipids from total plasma is markedly different from that of healthy subjects (9, 14-15), but the fatty acid profiles of these lipid fractions from the different lipoproteins have not been studied. It is therefore necessary to determine the distri-

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bution of lipid species among the different lipoproteins in hepatic disease. A non-homogeneous distribution would reflect an alteration in plasmatic lipoprotein metabolism.

All these data underline the influence of the functional state of the liver on the availability of energy-yielding substrates, and they indicate possible effects on the activity of cholesteryl ester transfer protein.

In the present work, to determine the influence of the liver function on the homeostasis of complex lipids in plasma, we have studied the lipoprotein profile and the fatty acid composition of lipids from VLDL, LDL and HDL, as well as from total plasma, in several groups of patients with hepatobiliary pathology.

the phospholipids, triacyclglycerols and cholesterol ester

were separated by thin layer chromatography, and their constituent Sy adds were methylated before analysis with 4 ml of su^hunc acid/methanol (1 + 2, by vol.) for 3 h at ^jSym^M quantitation of fatty acids was accomplished at 180 C m a Perkm Elmer 3920 Β gas liquid Chromatograph (Perkm Elmer Corp., Nor- walk, CT), equipped with a 2 m X 2 mm I. D. glass column filled with 5% of diethylene glycol succmate on gas chrom Q 100-120 mesh Quantitation was based on comparison of the relative re- sponses of the fatty acid methyl esters in the sample with the re- sponses of pure standards (Sigma, Chemical Co., Sigma-Aldnch Quimica S.A.).

Statistical analysis

Fatty acid methyl esters (percentages) from the different lipoproteins and plasma lipid fractions were determined for each disease and compared with those of the control group by the Newman Keuls Hartley test.

Materials and Methods

Subjects

Thirty-six patients of both sexes, between the ages of 23 and 72 years were studied. Twelve suffered from liver cirrhosis, in the majority of cases of ethylic origin, that was uncompensated in 7 and compensated in the rest. Fourteen had primary biliary cirrhosis, 8 in stages I and II and the remaining in HI. The evolution of the disease in the patients was from 1 to 10 years. Five patients presented other intrahepatic cholestasis distinct from primary biliary cirrhosis, 2 of scle- rotic cholangitis and 3 of a pharmacological origin. In the other intrahepatic cholestasis group, the evolution of the disease was 1 to 10 months. Five patients presented acute viral hepatitis, and were studied within 15 days of diagnosis. The age and sex distribution of the 6 healthy adults comprising the control group were similar to those of the patients. None was receiving medication or diet that could alter the lipid metabolism or the fatty acids. All samples for analysis were taken after fasting for 12 hours.

Isolation of lipoproteins and lipid analysis

Biood was withdrawn from patients and controls after a 12 hour period of fasting. Blood was collected over EDTA · Na2 (1 g/1) and rapidly centrifuged at 4 °C for plasma separation. Lipoproteins3) were isolated from a 10 ml plasma aliquot by sequential ultracentrifugation with a Beckman 50 Ti rotor in a Beckman L5-50 ultra- centrifuge (Beckman Instruments, Palo Alto, CA), as described (18), at the following density ranges: very low-density lipoproteins (VLDL), d < 1.006 kg/I; low-density lipoproteins (LDL), d 1.006 to 1.063 kg/1; high-density lipoproteins (HDL), d 1.063 to 1.21 kg/1. All salt solutions for density adjustment contained 1 mmol/1 EDTA · Na2. The lipoprotein-containing supematants were used for lipid analysis and determination of fatty acid profile without any further manipulation. Total cholesterol, free cholesterol, triacylglycerols and phosphatidylcholine were measured enzymatically in an autoanalyser Technicon RA-1000 (Technicon Ltd., Dublin, Ire- land). Esterified cholesterol was calculated as the difference between total and free cholesterol. Glycerol was measured in depro- teinized plasma (19) and this value was subtracted from those of triacyclglycerols in total plasma and in lipoprotein fractions. Total bilirubin, and albumin concentrations, as well as aspartate amino- transferase2), γ-glutamyl transferase2), and alkaline phosphatase2) activities were measured in serum in an Hitachi 747 autoanalyser.

Fatty acid profile analysis

Lipids were extracted from lipoprotein fractions and total plasma with chloroform/methanol (2 + 1, by vol.), (16). After extraction,

3) Lipoproteins VLDL, very low-density lipoproteins, (d < 0.006 kg/1); LDL, low-density lipoproteins, (d = 1.006-1.063 kg/1);

HDL, high-density lipoproteins, (d = 1.063-1.21 kg/1).

Results

Table 1 presents the general biochemical profile of the different groups of patients, and includes the results of the serum monitoring of aspartate aminotransferase, γ- glutamyl transferase, and alkaline phosphatase activities, and total bilirubin and albumin concentrations. In the profiles of the two cirrhotic patient subgroups, the hypo- albuminaemia and increase of bilirubin in the metaboli- cally uncompensated is notable, compared with the values for the compensated clinical state. The other biochemical quantities serving as indicators of the liver function were altered in the two subgroups, but with a greater statistical significance in the more seriously ill patients.

In the group of patients with intrahepatic cholestasis, there was a significant increase in the total bilirubin concentration, except in those included in subgroup primary biliary cirrhosis with primary, as yet incipient biliary cirrhois (stages I and II). The greatest alteration in the liver enzyme activities and increase of total bilirubin were found in the patients with primary biliary cirrhosis in stage III, compared with the other groups. The profile of those suffering from other intrahepatic cholestasis was the least altered in comparison with the controls.

The lipoprotein profiles of all the groups are summarized in table 2; those of the patients with primary biliary cirrhosis I and II are most similar to those of the controls. In the patients with liver cirrhosis (uncompensated liver cirrhosis and compensated liver cirrhosis), the most remarkable observation was the low levels of triacyclglycerols with normal levels of cholesterol in total plasma. In these cases, the lipoprotein profile showed a reduced number of VLDL particles, as indicated by the diminished content of both triacyclglycerols and cholesterol in this fraction, and the presence of LDL enriched with respect to triacylglycerols. In addition, HDL-cholesterol was decreased in the uncompensated liver cirrhosis group. In patients with acute viral hepatitis, and

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Arranz et al.: Composition of lipoprotein lipids in hepatobiliary diseases 703

Tab. 1 Hepatic function tests of patients with hepatobiliary dis- Serum samples were used for the evaluation of hepatic properties eases and controls. by routine analyses.

Uncompen- sated liver cirrhosis (n = 7)

Compen- sated liver cirrhosis

Primary biliary cirrhosis in stages I, II (n = 8)

Primary biliary cirrhosis in stage HI

Acute viral hepatitis

Other intrahepatic cholestasis (n = 5) (n = 5)

Controls

(n = 6) Aspartate amino-

transferase^a'^b (U/l) γ-Glutamyl transferasea-b (U/l)

97±74C 73 ±49° 118±29e 187±139C 931±637C 56±24C 18 ±8 132±78d 119 ±85 272±155d 372±284d 152±84d 64 ±27 23 ±18 Alkaline phos-

phatase^^b (U/l) Bilirubina-b

(μπιοΐ/ΐ) Albumin0· b (g/1)

329 ± 125C

97 ± 22e

26±3d

354 ± 298

32 ±21 38 ±2

875 ± 589C

43 ±39 -

2093 ±1691°

132±99C

-

427±168C

270±157d

-

316 ±223 108±82C

-

160 ±87 14±9

41±6

8 Means ± S.D.^dp < 0.01,^ep < 0.001.

b Significant differences between groups and control atc ρ < 0.05,

Tab. 2 Serum lipoprotein lipids of patients with hepatobiliary Plasma samples were subjected to sequential ultracentrifugation for diseases and controls. lipoprotein isolation, and the lipid composition was evaluated by

enzymatic analyses.

Uncompen- Compen- Primary sated liver sated liver biliary cirrhosis cirrhosis cirrhosis in

stages I, II (n = 7) (n = 5) (n = 8) Total cholesterol8* b 4.40 ± 1 .45 4.06 ± 1 .40 5.20 ± 0.98 (mmol/1)

Free cholesterol8·1» . 1.64 ±0.80 1.36 ±0.49 1.54 ±0.32 (mmol/1)

Esterified choles- 2.45 ± 0.78 2.72 ± 0.50 3.66 ± 1 .03 terola-b (mmol/1)

Fraction of esteri- 64 ±9 67 ±5 71±7 fied cholesterol8'0 (%)

Triacylglycerolsa'b 0.80±0.30d 0.65±0.11d 0.95 ± 0.29 (mmol/1)

LDL,cholesterola*b 3.40 ± 1.62 2.66 ± 1.09 3.50 ± 0.72 (mmol/1)

LDL-triacylglyc- 0.47 ± 0.22d 0.32 ± 0.09d 0.32 ±0.15 erolsa*b (mmol/1)

HDL-choles- 0.67±0.86^d 1.14 ±0.49 1.37 ±0.57 terola-b (mmol/1)

HDL-triacylglyc- 0.09 ±0.06 0.13 ±0.05 0.14 ±0.05 erolsa-b (mmol/1)

VLDL-cholestes- 0.08 £ 0.05e 0.05 ± 0.03e 0. 1 6 ± 0. 13 terola-b (mmol/1)

VLDL-triacylglyc- 0.14±0.08e 0.14±0.10C 0.29 ±0.16 erolsa'b (mmoi/1)

Primary Acute Other Controls biliary viral intrahepatic

cirrhosis in hepatitis cholestasis stage III

(n = 6) (n = 5) (n = 5) (n = 6) 7.84±3.48C 5.30 ±1.80 4.81 ±0.50 4.89 ±1.18

3.56 ±3.22 2.28±0.65^C 1.72 ±0.88 1.36 ±0.41 4.25 ±1.36 2.83 ±1.86 3.10 ±1.78 3.46 ±0.93

63±25 52±19C 44±16 72±4

1.50 ±0.82 3.06±0.87e 2.58 ±1.53 1.15 ±0.23 6.00 ±3.43 3.94 ±1.80 3.56 ±1.64 3.37 ±0.70

0.58 ± 0.48 1 .76 ± 0.64^e 0.82 ± 0.53 0. 1 5 ± 0.03 1.55 ±0.49 0.72±0.28C 0.49±0.18d 1.09 ±0.21 0.25±0.10C 0.17 ±0.06 0.11 ±0.05 0.11 ±0.02

0.30 ±0.23 0.49±0.01^d 0.65 ±0.72 0.23 ±0.03 0.62 ±0.50 0.83 ±0.45 1.11 ±0.39 0.64 ±0.1 7

a Mean ± S.D. dp < 0.01, ep < 0.001.

b Significant differences between groups and control atc ρ < 0.05,

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less notably in patients with other intrahepatic cholestasis, the triacyclglycerol concentration in total plasma was markedly elevated, whereas that of cholesterol was within the normal range. This corresponded to an eleva- tion of VLDL of normal composition and the presence of LDL enriched with respect to triacyclglycerols. In these two groups, the HDL-cholesterol concentration was significantly lower than in controls. The patients with primary biliary cirrhosis in stage III showed no apparent change in the lipoprotein triacylglycerol content, but showed major alterations in cholesterol metabolism, as indicated by the increase in cholesterol concentration in total plasma and in LDL. This increase corresponded mainly to free cholesterol. A similar situation was found in patients with acute viral hepatitis, which probably reflects the deficiency of lecithin : cholesterol acyltransferase activity characteristic of these diseases (6).

The percentages of the fatty acids4)-myristic (C14), palmitic (C16), palmitoleic (C16: 1), stearic (CIS), oleic (CIS : 1), linoleic (CIS : 2), eicosatrienoic (C20 : 3ω6), arachidonic (C20 :4) acids — in lipoprotein and total plasma triacylglycerols, cholersteryl esters and phospholipids are summarized in tables 3, 4 and 5, respectively.

In each group of patients the fatty acid composition of triacylglycerols, cholesteryl esters and phospholipids was very similar for VLDL, LDL, HDL and total plasma, but there were significant differences between each group of patients and between patients and the control group. Generally, the cholesteryl ester fraction was the most sensitive, showing differences in fatty acid composition in all the patient groups with respect to controls, whereas the changes were more moderate in the phospholipids. When all the patient groups are consid- ered together, it appears that the content of the polyunsaturated CIS : 2 and C20 :4 is lower than in controls, a change that is compensated by moderate increases in the other fatty acids. The most important changes were observed in patients with liver cirrhosis, especially in those with uncompensated liver cirrhosis, where the linoleic acid content of total plasma cholesteryl esters was 41.6%, compared with 58.3% in the controls (p < 0.001) (tab. 4). The corresponding values for arachidonic acid were 3.2 and 4.6%, respectively (p < 0.05) (tab. 4). Among the other groups of patients, acute viral hepatitis was the most similar to the uncompensated liver cirrhosis, and therefore different from the controls. Patients with other intrahepatic cholestasis also showed significantly lower contents of linoleic acid in the three lipid fractions of lipoproteins and total plasma than the control group. The patients with primary biliary cirrhosis had a more dispersed fatty acid profile, and the

4) Fatty acids are designated by the number of carbon atoms fol- lowed by the number of double bonds.

differences from the control group were less significant.

Finally, more moderate differences were found in the subgroup of primary biliary cirrhosis I and II.

Discussion

This study documents the lipoprotein profile and the fatty acid composition of triacylglycerols, cholesteryl esters and phopsholipids from VLDL, LDL, HDL and total plasma in patients suffering from hepatic disease, with distinct types of intrahepatic ch'olestasis, as compared with control subjects. The main observation of the present study is that in patients with hepatobiliary disease, despite the profound changes in the lipoprotein profile and the lower abundance of polyunsaturated fatty acids in complex lipids, the fatty acid composition of all triacylglycerols, cholesteryl esters and phospholipids was very similar among the different lipoproteins and in control subjects. This indicates that in the controls, as well as in the studied patients, the cholesteryl ester transfer protein-promoted exchange of lipids between plasmatic lipoproteins is very rapid, resulting in an homogeneous distribution of lipid species between lipoprotein particles.

Apart from the liver, other tissues also synthesize cholesteryl ester transfer protein mRNA and secrete active protein (21, 22), so that plasma cholesteryl ester transfer protein may be derived from more than one source. In this context, it is worth mentioning that in chronic hepatitis, there is a marked decrease in plasma lecithin : cholesterol acyl transferase activity, an enzyme of exclusive hepatic origin, whereas plasma cholesteryl ester transfer protein activity and mass remain stable (6), which indicates that liver damage is not necessarily ac- companied by changes in plasma lipid transfer activity.

Therefore, it is veiy likely that the cirrhotic patients studied in this work had a normal cholesteryl ester transfer protein activity, so that the velocity of the inter- change of neutral lipids between the different classes of lipoproteins was unaffected.

We consider next the difference in fatty acid composition between the complex lipids in cirrhotic patients and those in normal individuals. In general, a diminution of the polyunsaturated fatty acids CIS : 2 and C20 : 4 was observed, which was more pronounced in the patients with uncompensated liver cirrhosis or acute viral hepatitis. Compared with the controls, the triacylglycerols of these patients contain in smaller percentage of CIS : 2, the phopholipids are lower in C20 :4, while both fatty acids are decreased in the cholesteryl esters. These lower levels are compensated by a slight increase in the rest of the fatty acids. We should point out that this fatty acid profile, both in the control group and in the patients, is similar to that found by other authors in population groups in our geographical area (16, 17, 23). The com-

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position of fatty acids in cirrhotic patients resembles that found in situations of linoleic acid deficiency (24-27).

It is known that patients with hepatobiliary disease suf- fer frequently from an anorexic, emetic and nauseous state, which in chronic situation can lead to severe malnutrition (28). In fact, the anthropometric estimation (29) of the two cirrhotic subgroups (data not shown) indicated that 50% of the uncompensated were clearly undernourished, while the rest were poorly nourished.

Also, these data agree with the lower percentage of linoleic acid in triacylglycerols, which is used to evaluate

the content of this fatty acid in the dietary fat (30). The compensated cirrhotic patient presumably had a better nutritional state, but this quantity fluctuated more in this subgroup, possibly reflecting a greater alimentary heterogeneity and subjacent liver damage, in spite of the fact that this clinical state is more benign.

In patients with intrahepatic cholestasis, acute viral hepatitis and other intrahepatic cholestasis, we observed that the relative content of linoleic acid was decreased in parallel with the degree of cholestasis and the severity of the hepatic disease. In the subgroups primary biliary

Tab. 3 Fatty acid composition of triacylglycerols from total plasma and lipoproteins in patients with hepatobiliary diseases and controls.

Plasma samples were subjected to sequential ultracentrifugation for lipoprotein isolation. The lipids were extracted and separated by TLC and the fatty acid composition was evaluated as methyl esters by GLC.

Compen- sated liver cirrhosis (n = 5)

Primary biliary cirrhosis in stage III (n = 6)

Acute viral hepatitis (n = 5)

Other intrahepatic cholestasis (n = 5)

Controls

(n = 6) Myristic acid C14 : 0 (%)a

VLDL 1.3 ±0.2 LDL 1.3 ±0.2 HDL 2.3 ±1.8 Total plasma 1.8 ±0.7 Palmitic acid C16:0(%)a

VLDLLDL HDLTotal plasma

28.4 ±3.8 25.5 ± 3.9 25.7 ± 5.5 27.3 ± 4.6 Palmitoleic acid C16 : 1 (%)a

VLDL 4.1 ±0.9 LDL 4.8 ±1.2 HDL 5.2 ± 2.0 Total plasma 5.1 ± 1.7 StearicacidC18:0(%)a

VLDL 6.6 ± 0.7e

LDL 5.8 ± 0.9e

HDL 6.2 ± 0.5C

Total plasma 5.3 ± 0.9e

Oleic acid CIS: 1 (%)a

VLDL 47.3 ± 2.5 LDL 50.8 ± 4.7 HDL 46.0 ± 6.7 Total plasma 50.2 ± 3.8 Linoleic acid C18 : 2 (%)a

VLDL 10.9±2.5e

LDL 10.4±2.6C

HDL 11.6±5.5e

Total plasma 9.6±3.0e

2.4±1.0d

1.1 ±0.2 1.6 ±0.4 1.6±0.1

26.2 ± 2.0 24.8 ±3.1 25.0 ±1.7 25.8 ± 2.2

4.0 ±1.2 4.0 ±1.6 4.5 ±1.5 4.7 ±1.0

5.8±1.2C

4.5 ±1.8 6.4 ±2.8 4.3 ±1.6

45.2 ±4.1 50.2 ±4.9 48.5 ± 6.6 49.3 ± 3.4

15.6 ±12.6 14.4 ±7.13 12.4±4.6d

13.0±3.0d

1.7±0.4C

1.3 ±0.5 1.4 ±0.6 1.2 ±0.4

26.3 ±4.5 25.4 ± 4.9 24.9 ±4.7 26.9 ± 6.8

4.6 ±1.4 3.9 ±1.4 4.7 ±1.9 4.6 ±1.8

4.0 ±1.1 4.9 ± 0.7C

4.6 ±1.3 4.7±1.0C

40.8 ± 4.3 43.0 ±4.8 43.2 ± 3.5 42.9 ± 6.0

21.8 ±7.2 20.4 ± 7.3 20.2 ± 6.8 19.1 ±7.7

1.8 ±0.9 1.9±1.1 2.2 ±1.6 2.0 ±0.9

25.3 ±1.8 25.0 ±3.3 25.1 ±4.5 26.3 ± 4.5

4.2 ±1.4 4.6 ±3.7 3.7 ±1.9 5.1 ±3.0

3.7 ±1.4 4.2 ±1.3 7.6 ± 0.5 3.7 ± 0.8C

41.2 ±7.9 39.8 ± 7.3 35.4±4.2C

40.0 ±5.8

22.0 ± 9.4 22.4 ±8.9 23.8 ±8.8 21.0 ±10.5

2.1 ±1.6 2.6 ±2.4 2.3 ±1.1 2.4 ±1.3

27.1 ±3.5C

28.1±4.6C

26.9 ± 3.2 27.5 ± 3.8

5.2 ±3.0 6.0±1.8C

4.8 ±2.7 4.8 ±2.0

5.4 ± 2.3C

6.2 ± 2.5 6.1 ±0.8°

5.5 ±1.9

42.3 ±4.1 42.3 ±4.1 40.8 ± 8.9 40.2 ± 7.5 18.05±5.4C

14.3 ±6.2 17.5±3.7C

17.7 ±5.6

1.9±0.7C

1.3 ±0.6 1.3 ±0.7 1.8 ±0.4

27.2 ± 3.3 24.1 ±4.3 24.2 ± 5.4 26.8 ± 4.5

5.0 ± 2.4 4.0 ±1.8 4.9 ± 2.8 4.6 ±3.0

3.0 ±0.8 3.7 ±0.5 4.6 ±1.0 3.8 ± 0.9

44.6 ±6.1 48.6 ± 6.5 47.2 ±7.1 46.7 ± 4^C

17.5 ±5.7 16.9 ±5.9 17.9 ±6.8 16.2 ±4.6

1.1 ±0.2 0.8 ± 0.2 1.0 ±0.4 1.2 ±0.4

22.9 ± 2.6 21.9 ±2.3 22.8 ± 2.0 23.7 ± 2.4

4.0 ± 0.4 3.5 ±0.4 4.0 ± 0.4 4.3 ± 0.2

3.0 ±0.8 3.6 ±0.3 4.3 ±1.0 2.7 ± 0.2

44.1 ±3.3 44.6 ± 3.6 43.2 ±4.1 43.2 ±1.8

25.7 ± 3.4 24.5 ± 5.5 23.4 ±4.8 24.2 ± 3.6 Arachidonic acid C20 : 4 (%)a

VLDLLDL HDLTotal plasma

1.3 ±1.3 1.4 ±0.9 2.2 ±1.8 1.5 ±0.4

1.4±1.1 0.9 ± 0.3 1.4±1.3 1.3 ±0.4

0.6 ± 0.5 0.9 ±1.1 0.8 ± 0.7 0.5 ± 0.3

1.1 ±0.5 1.3±1.1 l.7±1.8 1.3 ±1.0

1.3 ±0.9 1.2±1.1 i.2±0.7 1.1 ±0.8

0.77 ± 0.2 1.2 ±0.5 1.2 ±1.0 0.5 ± 0.2

1.0 ±0.5 1.3 ±0.5 1.0 ±0.5 0.7 ± 0.2

a Values are means ± S.D. Significance differences between groups and control at cp < 0.05,

dp < 0.01, ep < 0.001.

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cirrhosis II and primary biliary cirrhosis in stage III, however, the percentage of linoleic acid was maintained within the control group range, although with rather high interindividual dispersion. The fact that these patients with primary biliary cirrhosis did not lose appetite even in severe jaundice, (31) together with the diet in polyunsaturated fats they were recommended to take for their chronic hypercholesterolaemia (32), probably con- tribute to the unaltered content of linoleic acid observed in primary biliary cirrhosis in stages I and II and primary biliary cirrhosis in stage III patients.

The primary role of the liver in the synthesis of arachidonic acid is reinforced in the present study, which clearly documents the low content of this fatty acid in patients with hepatocellular damage. Interestingly, these data contrast with the increase of arachidonic acid in cystic fibrosis without liver affectation (29), probably as a compensating effect of the diminution of its metabolic precursor - linoleic acid - which is characteristic of this disease (33).

The changes observed in the lipoproteins deserve some comment. Patients with severe choiestasis, acute viral

Tab. 4 Fatty acid composition of cholesteryl esters from total plasma and lipoproteins in patients with hepatobiliary diseases and controls.

Plasma samples were subjected to ultracentrifugation for lipoprotein isolation. The lipids were extracted and separated by TLC and the fatty acid composition was evaluated as methyl esters by GLC.

Other intrahepatic choiestasis (n = 5)

Controls

(n = 6) Myristic acid C14 : 0 (%)a

VLDL 1.5 ±0.1 LDL 1.0 ±0.1 HDL 1.3 ±0.7 Total plasma 0.9 ± 0.3C

Palmitic acid C16 : 0 (%)^a VLDLLDL

HDL Total plasma

15.0±1.0^e 14.3±1.5^d 15.0±2.0e

15.0 ± 0.9^d Palmitoleic acid C16 : 1 (%)a

VLDL 5.0 ± 0.9

LDL 5.7±1.6^C

HDL 6.5 ± 2.2^d

Total plasma 5.3 ± 0.9 StearicacidC18:0(%)^a

VLDL 2.6 ± 0.7

LDL 2.7 ± 0.2d

HDL 3.2±1.5^d

Total plasma 2.2 ± 0.9^C 01eicacidC18: 1 (%)

VLDL 33.6 ± 4.7^C

LDL 33.2 ± 6.8d

HDL 33.2 ± 4.4d

Total plasma 32.1 ±5.1d

Linoleic acid CIS: 2 (%)a

0.7 ± 0.2 0.7 ± 0.5 0.5 ± 0.3 1.0±0.3^C

13.7 ±3.0 15.6 ±5.0 15.8 ±5.3 14.4 ±3.6

3.3 ±1.0 3.1 ±1.9 3.6 ±1.9 3.2 ±1.6

2.4 ± 0.9 3.1 ±2.5 3.2 ±2.2 2.8 ±1.7 27.2 ± 6.0 26.3 ± 7.5 26.2 ± 7.7 25.9 ± 5.0

0.5 ± 0.4 0.5 ±0.1 0.5 ± 0.3 0.5 ± 0.3

12.4±1.8^C 12.5 ±1.5 12.1 ±1.6 12.7 ±2.6

3.6 ±3.7 3.5 ±2.5 3.7 ±4.6 4.1 ±3.8

0.9 ± 0.5 0.8 ± 0.3 1.0 ±0.2 0.9 ± 0.3

23.7 ± 7.7 21.1 ±4.9 21.0 ±5.7 21.5 ±5.8

0.5 ± 0.2 0.5 ±0.1 0.7 ±0.3 0.6 ± 0.2

11.8±1.5 11.0 ±0.8 12.5 ±1.0

7.9±4.6C

7.5 ±5.5 6.1 ±4.0 6.2 ±4.9

0.8 ± 0.3 0.8 ±0.5 1.1 ±0.2 0.9 ± 0.4

20.5 ± 4.0 19.8 ±5.4 15.3 ±2.9 20.5 ± 5.0

2.0 ±1.5 1.0 ±0.7 2.6±1.2C

1.8±0.5^C

17.2±4.3^C 13.9±1.2C

16.6±4.4^C 15.8±1.6^C

7.7±5.1^C 8.4 ± 4.6C

6.9 ± 3.8 8.1±4.3C

3.4:1.8:

3.7:2.4:

23.8:

22.7:

24.0:

23.8:

:0.8:2.2^C :0.4C

:6.5 :5.5:4.3 :5.6 LDLVLDL

HDL Total plasma Arachidonic acid VLDLLDL

HDL Total plasma

39.9 ± 8.3^C 41.5±8.1d

37.0 ± 7.2e

41.6±4.2^C C20 : 4 (%)^a

3.0 ± 1.2^C 3.6 ± 1.3d

4.1±1.7^e 3.2±1.5^C

52.3 ± 9.0 46.4 ±12.7 45.7 ±13.5 49.1 ±11. 4

3.8 ±1.0 4.7±1.4^C 4.3±1.4^d 4.0 ± 1.0C

56.9 ±12.2 55.5 ± 9.9 55.1 ±11.0 56.9 ±13.2

5.2 ±2.7 5.7 ±2.0 6.3 ±2.4 5.3 ±1.2

48.9 ± 7.9 54.7 ± 9.2 58.6 ± 6.4 53.2 ± 8.7

5.2 ± 2.3 6.2 ±2.1 6.3 ±1.6 5.7+1.7

36.1±8.2^d 42 7 + 6 8^C 37.6 ±11. 3' 42.2 ± 3.9d

3.9 ± 0.5^C 5.2±1.0C

5.1 ±1.2^C 3.8 + 0.6C

1.4 ±1.7 0.9 ± 0.4 0.8 ± 0.3 0.8 ± 0.3^C

14.6 ± 3.2°

14.3 ±2.5 12.7 ±1.9 14.5 ±2.0

6.2 ±4.0 4.7 ±3.7 3.9 ±4.1 6.1 ±4.3

2.4 ±1.5 1.7±1.0 2.1 ±1.0 1.2 ±0.3

26.7 ± 4.4 27.8±4.7^d 25.7 ± 3.5^C 26.6 ± 3.lc

42.3 ± 7.2^C 44.2 ± 7.8C

46.5 ± 6.7C

45.6 ± 7.8^C

4.7 ±1.0 5.5 ±1.8 6.0 ±1.5 4.9 ± 2.3

0.7 ± 0.4 0.7 ± 0.5 0.5 ±0.1 0.5 ±0.1

10.1 ±0.5 11.2±1.0 10.7 ±0.6 11.7 ±0.6

3.2 ±1.6 2.8 ±1.0 2.5 ± 0.5 2.5 ± 0.4

1.7 ±0.9 1.5 ±1.6 1.0 ±0.2 0.9 ± 0.2

22.5 ±4.5 18.8 ±2.8 18.4 ±3.2 20.7 ± 2

54.4 ± 6.0 57.7 ± 4.7 58.9 ±3.0 58.3 ±3.7

6.4 ±1.6 7.3 ±1.3 8.1 ±1.1 4.6 ± 0.6

vaiuto aic invalid -l. O dp < 0.01, cp < 0.001.

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hepatitis and primary biliary cirrhosis III, showed increased concentrations of free cholesterol, which were probably related to the well-known decreased activity of lecithin : cholesterol acyltransferase in this pathologic state (5-7). In the acute viral hepatitis group, increased levels of triacylglycerols were observed in agreement with previous results (34). In contrast, patients with liver

cirrhosis had decreased plasma levels of triacylglycerols, mainly due to a marked decrease in the number of VLDL particles. We found in previous work that VLDL from cirrhotic patients had an altered apolipoprotein composition, with a very low content of apolipoprotein E (34-36), which probably indicates a reduced pro- duction of VLDL by the liver in this disease. In addition, Tab. 5 Fatty acid composition of phospholipids from total plasma

and lipoproteins in patients with hepatobiliary diseases and controls.

Plasma samples were subjected to sequential ultracentrifugation for

lipoprotein isolation. The lipids were extracted and separated by TLC and the fatty acid composition was evaluated as methyl esters by GLC.

Other intrahepatic cholestasis (n = 5)

Controls

(n = 6) Myristic acid C14 : 0 (%)^a

VLDL 0.4 ± 0.2 LDL 0.8 ± 0.4

HDL 1.1 ±0.8

Total plasma 0.7 ± 0.3C

Palmitic acid C16 : 0 (%)a

VLDLLDL Total plasmaHDL

31.5±1.8 32.8 ±2.0 28.4 ± 2.2 31.1 ±1.9 Palmitoleic acid C16 : 1 (%)^a VLDL 1.4 ±0.6

LDL 1.8±0.8C

HDL 1.8 ±0.6

Total plasma 1.5±0.6^C StearicacidC18:0(%)^a

VLDL . 16.3 ±0.4

LDL 14.0 ±3.1

HDL 14.1 ±3.2

Total plasma 15.3 ±0.8 01eicacidC18:l (%)^a

VLDL 18.3±1.8d

LDL 20.2 ± 3.8d

HDL 21.9±2.7e

Total plasma 19.5±2.5e

LinoleicacidC18:2(%)a

VLDL 23.5 ±1.2

LDL 21.4 ±2.7

HDL 21.5 ±4.5

Total plasma 22.8 ±3.1 Eicosatrienoic acid C20 : 3ω6 (%)a

VLDL 1.8 ±1.2

LDL 2.5 ±1.7

HDL 2.4 ±1.6

Total plasma 1.9 ±0.9

0.3 ±0.1 0.5 ±0.1 0.3 ±0.1 0.5 ±0.1

32.0 ± 6.2 32.7 ±3.4 32.6 ± 6.8 32.9 ± 3.6

1.9±0.4C

1.3 ±0.9 1.5±0.7C

1.8±0.2^d

14.0 ±3.6 13.7 ±3.4 12.8 ± 5.0 14.0 ±2.8

21.9 ±8.4 21.6 ±8.6 20.9 ±6.1 20.7 ± 4.4

22.1 ±7.4 23.1 ±7.2 23.8 ± 9.7 21.6 ±5.7

2.1 ±0.6 1.5 ±0.9 1.5±l.l^c 1.9 ±0.5

0.4 ±0.1 0.4 ±0.1 0.4 ±0.1 0.3 ±0.1

30.8 ± 3.9 34.6 ±4.1 32.3 ±3.8 33.1 ±3.4

1.2 ±0.5 0.7 ± 0.6 1.0 ±0.8 0.6 ± 0.5

16.7 ±2.8 16.1 ±2.5 15.9 ±1.7 16.9 ±2.6

13.9 ±2.2 12.3 ±3.3 12.8 ±3.0 12.9 ±2.1

25.2 ± 6.8 24.9 ± 5.8 24.6 ± 6.5 25.9 ± 8.3

2.1 ±1.2 2.4 ±1.9 2.7 ±1.6 2.4 ±1.6

0.6 ± 0.3 0.4 ±0.1 0.5 ± 0.3 0.7 ± 0.4

30.1 ±3.3 35.6 ±4.8 31.4 ±4.9 32.5 ±4.4

1.1 ±0.8 1.9 ±1.2 1.0 ±0.7

16.8 ±2.0 16.3 ±5.4 15.7 ±2.5 15.2 ±2.9

14.2 ±4.4 13.0 ±5.3 12.0 ±4.1 12.2 ±3.3

25.9 ± 7.2 32.1 ±7.8 26.4 ± 6.8 25.2 ± 5.0

2.2 ± 0.9 2.4 ± 0.8 2.5 ± 0.6 2.7 ±1.1

1.1 ±0.9 0.5 ± 0.3 0.5 ± 0.2 0.5 ±0.3

31.2 ±2.0 33.1 ±2.6 30.3 ±1.4 31.8 ±2.0

1.9 ±1.4 0.9 ± 0.4 1.2±0.5C

1.3 ±'0.5

17.2±0.9^C 16.5 ±0.7 16.8 ±1.2 16.0 ±0.4

17.2 ±3.0°

15.2 ±3.9 15.8±3.1^C 16.1 ±3.4

18.8±2.0C

20.0 ± 2.7 20.6 ±2.1 21.0 ±2.1

2.4 ±1.2 3.1 ±1.3 3.5 ±1.2 3.2 ±0.9

0.3 ±0.1 0.4 ± 0.3 0.9 ± 0.5C

0.4 ± 0.2

31.7±4.3 32.1 ±2.7 32.0 ±4.8 32.9 ± 2.5

1.0 ±0.6 0.9 ± 0.7 0.9 ± 0.5 1.3 ±0.9

17.8 ±3.3 16.9 ±2.8 16.9 ±2.5 17.2 ±2.1

14.8 ±4.3 14.5 ±4.3 14.5 ± 2.2 15.4 ±2.4

21.5 ±4.6 21.7 ±2.4 20.4 ±3.5 20.1 ±3.8

4.2 ±1.1 4.3 ±1.6 4.9 ± 0.6 4.1 ± 1.5C

0.5 ± 0.2 0.4 ±0.1 0.3 ±0.1 0.3 ±0.1

30.4 ±3.1 33.8 ±3.4 30.3 ±1.6 34.3 ± 2.5

0.9 ±0.2 0.7 ± 0.3 0.7 ± 0.2 0.8 ± 0.2

16.1 ±0.8 15.6 ±0.7 16.3 ±0.9 16.5 ±1.2

12.8 ±1.9 12.8 ±1.9 11.9±1.9 12.9 ±1.9

24.3 ± 2.2 22.8 ±1.8 22.9 ±1.9 23.0 ±1.5

3.0±1.1 3.1 ±1.5 3.1 ±0.7 2.3 ± 0.6 Arachidonic acid C20 : 4 (%)a

VLDLLDL Total plasmaHDL

6.7 ± 2.5C

6.2±3.2C

7.6±2.8C

7.0±2.2C

5.1±3.4d

5.5±2.1d

5.9±1.5d

6.6 ± 2.6C

9.6 ±3.6 7.9 ±2.3 9.9 ±3.3 8.7 ±1.9

8.9±1.6C

7.9 ± 2.2 9.8 ± 2.6 9.0 ± 2.9

9.2±l.lc

9.0 ±1.4 10.8 ±1.3 9.3 ±1.9

9.0 ±2.1 8.9 ±1.9 9.7 ± 2.5 8.4 ±2.7

12.1 ±2.1 11.3 ±2.4 12.5 ±3.1 10.2 ±1.8

a Values are means ± S. D. Significance differences between groups and control atc ρ < 0.05,

dp < 0.01, cp < 0.001.

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we observed that the LDL-triacylglycerol concentration is increased in these patients as compared with controls.

Actually, LDL from these patients appear to be enriched with respect to triacylglycerols because of the elevated LDL triacylglycerol cholesterol mass ratio. No Lp-X was detected in the LDL fraction by agarose electropho- resis (data not shown). Whether the decreased unsatura- tion of fatty acids in the lipids of these lipoproteins con- tributes directly to the altered metabolism of LDL in these patients deserves further investigation.

In conclusion, in a broad group of patients with liver cirrhosis or other related pathologies, we demonstrated profound changes in plasma lipid levels and lipoprotein distribution, and a diminished content of linoleic acid as indicative of the undernourished state of some of these patients. Despite these alterations, triacylglycerols and cholesteryl esters were homogeneously distributed

among the different plasma lipoproteins in cirrhotic patients as well as in normal individuals. This indicates that the plasma cholesteryl ester transfer protein activity present in these conditions is high enough to promote a rapid exchange of these lipids between the lipoproteins and it demonstrates the possible importance of extrahepatic synthesis of cholesteryl ester transfer protein.

Acknowledgements r

We thank Dr. A. Cano of the Gastroenterology Service, Hospital Ramon y Cajal for patient care. We thank Drs. J. J. Arias and J.

Calabuig from Ambulatorio San Bias and Dr. C. Grande and Dr.

Olveira of the Hospital of La Paz for helping to find HAV patients.

We thank A. Reviriego, Carmen Coello and Maria Cruz Botas for technical assistance, and Shirley McGrath for linguistic assistance.

This work was supported in part by the Fondo de Investigaciones Sanitarias (94/0484), Spain.

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Received April 20/Sepiember 4. 1995/February 51 1996 Corresponding author: Maria Isabel Arranz Pena, Servicio de Bioquimica Clinica, Hospital Ramon y Cajal, E-28034 Madrid, Spain

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